Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.
Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.
Despite a concentrated effort by health organizations, tobacco product use remains the leading cause of preventable death worldwide, with the majority of these deaths attributed to cigarette smoking1. Since entering the market in 2003, electronic cigarettes have been growing in popularity among tobacco product users. Currently, electronic cigarettes are the most popular alternative to conventional cigarettes among American adults (~5%)2 and the most popular nicotine delivery system among middle (~5.3%) and high schoolers (~16%)3. If current trends continue, electronic cigarettes can be expected to replace conventional cigarettes for future generations. However, the health consequences of electronic cigarette use remain unclear.
Research on electronic cigarettes did not start in earnest until electronic cigarette popularity rapidly increased in 20133,4. Since that time, a number of different models have been employed to address the question of their toxicity. However, the results of many studies are conflicting, and while it seems that electronic cigarettes are generally less toxic than conventional cigarettes there is no current consensus on the health consequences of electronic cigarette use5,6,7. Our previous research indicates that electronic cigarettes are significantly less toxic to the vascular endothelium than conventional cigarettes, despite their ability to cause DNA damage and the induction of oxidative stress and cell death8. However, more research is necessary before we can draw firm conclusions about the health consequences of electronic cigarette use.
As conventional cigarettes are a leading cause of preventable vascular disease9, there is a growing interest in the vascular health risk of electronic cigarette use10,11,12. In order to study the effects of electronic cigarettes on the vascular system, our lab developed a microcontroller operated smoking/vaping device (Figure 1)8. This device is capable of generating liquid extracts of either conventional cigarette smoke or electronic cigarette aerosol in either aqueous or organic solvents. As airflow is controlled by the combination of an adjustable air flow regulator and a PBASIC timing program, the device can be used to generate extracts according to any number of user defined protocols. Here we detail the assembly and operation of this device as well as two potential applications: in vitro cell viability assessment and gas-chromatography mass-spectrometry.
Figure 1: Smoking/Vaping Device. Schematic for the physical assembly of the smoking/vaping device in both the cigarette/cigarette like electronic cigarette (e-cig) configuration (A) and the tank electronic cigarette configuration (B). Component Key: 1) Inhalation port; 2) primary collection impinger; 3) overflow impinger; 4) Buchner flask vacuum trap; 5) normally open solenoid valve; 6) BS1 microcontroller; 7) air flow regulator; 8) 510 threaded electronic cigarette tank base. Please click here to view a larger version of this figure.
1. Assembly of the Device
Figure 2: Electrical Schematic and PBASIC Code. Figure 2A displays the electrical schematic for assembling the electrical circuit necessary to activate both the normally open solenoid valve and the heating coil of button activated electronic cigarettes (through the 510 threaded electronic cigarette tank base). The electrical parameters of the heating coil (P: Power; R: Resistance; and I: Current) are projected and should be empirically verified with a multimeter post assembly. Figure 2B displays the PBASIC timing program needed to control the circuit in Figure 2A (also available at https://github.com/ChastainAnderson/SVL). The timing constants SVT & IPT (#5 & #6) are in units of ms and are set to provide an activation time of 2 seconds and a downtime of 28 s. Please click here to view a larger version of this figure.
2. Sample Storage and Preparation
3. General Operation of Cigarette Smoke/Electronic Cigarette Aerosol Extraction Device
4. Filtration and Storage
5. Cleaning the Device
6. Sample Application 1: Neutral Red Uptake Cell Viability Assay
7. Sample Application 2: Gas Chromatography Mass Spectrometry
Within 24 hours of the exposure of human umbilical vein endothelial cells to either conventional cigarette smoke extract (CSE) or electronic cigarette aerosol extract (EAE), there is a significant (control vs. CSE P <0.001; control vs. EAE P <0.01; n = 6) reduction in cell viability (Figure 3A). Extracts were generated with a puffing profile of 2, 2 second, 55 mL puffs per minute and normalized based on molar concentration of nicotine consumed by the device. Exposure to 500 µM consumed nicotine equivalents of CSE dramatically reduces viable cells to 11.06 ± 0.28% of control, and exposure to 500 µM consumed nicotine equivalents of EAE reduces viable cells to 86.65 ± 4.60% of control.
Figure 3B demonstrates the volatility based separation of electronic cigarette components from a commercial electronic cigarette by gas chromatography. The components were then identified via quadrupole mass spectrometry. Identified components, in order of volatility, include: propylene glycol, acetyl propionyl, chlorobutanol, glycerol, nicotine, and 3-nitropthalic acid. Of these only propylene glycol, glycerol, and nicotine were disclosed on the product label1.
Figure 3: Sample Applications: Cell Viability and GC-MS. Figure 3A displays the results of a neutral red uptake assay performed on human umbilical vein endothelial cells exposed to 500 µM consumed nicotine equivalents of either conventional cigarette smoke from a 3R4F research reference cigarette (CSE) or electronic cigarette aerosol from a commercially available electronic cigarette (EAE). Bars are mean +/- standard deviation. Significance determined by two tailed, unpaired, t-test and results indicated by asterisks: ** P <0.01; *** P <0.001; n = 6. Figure 3B displays the results of a gas chromatograph of electronic cigarette aerosol solubilized in acetone. Peaks represent individual compounds ordered by retention time (volatility) and were identified by quadrupole mass spectrometry. 1) propylene glycol; 2) acetyl propionyl; 3) chlorobutanol; 4) glycerol; 5) nicotine; 6) 3-nitropthalic acid. Please click here to view a larger version of this figure.
The most critical elements of this protocol are ensuring the device is clean at the start and finish of each extraction, and ensuring that all seals are maintained so that air flow remains consistent. If the device is not properly cleaned, there is a risk of carry over between samples. Additionally, if the device is left unclean for an extended period of time condensed aerosol and dried solvent can block the system. Note that it is normal for there to be a pressure drop when puffing a conventional cigarette and the airflow meter should be adjusted to provide the desired airflow during the puff, not while the device is pulling room air. A key feature of this method is the ability to be adapted to keep up with the advancement of electronic cigarette technology. For instance, many electronic cigarettes require button press activation of the heating coil. This device directly incorporates the heating coil into the control circuit (Figure 2A) mimicking a button press at user programmed intervals. The primary limitations of this method arise from the lack of well characterized standard operating procedures for electronic cigarette use. While we can use a research reference cigarette14 and international protocols15,16 for conventional cigarettes, we are merely adapting these methods to electronic cigarettes and cannot guarantee that it appropriately models electronic cigarette user behavior. Additionally, this protocol produces extract in a liquid medium. While this is appropriate for certain cell types, such as endothelial cells, other cell types, such as airway cells, may be better studied through direct exposure to electronic cigarette aerosol.
The nature of this device allows it to be updated as new standard operating procedures are developed. Several points of modification present themselves that could allow for the device to be tailored to specific questions. Newer electronic cigarettes encompass a higher range of wattage than earlier models of electronic cigarettes17. In the schematic presented in Figure 2A, both the resistor adjacent to the heating coil and the heating coil itself could be swapped for components with different resistance values (or even variable resistance) to modulate the final power used to aerosolize the electronic cigarette liquid. Final theoretical power in the atomizer can be calculated with the conventional power equations:
or
where P: Power; V: Voltage; R: Resistance; and I: Current.
As there is no broadly accepted international standard operating procedure for electronic cigarette use, and different groups may wish to employ different parameters and puffing profiles. A common electronic cigarette standard is CORESTA CRM8118, though some groups continue to use modified versions of conventional cigarette smoking protocols such as ISO 3088:201215 and WHO TobLabNet SOP 116. Additionally, many laboratories continue to use laboratory and/or institution specific regimes. In this instance, we employed a square wave puff profile consisting of 2, 2 second, 55 mL puffs per minute; however, the modular, programmable nature of the device allows it to be adapted to other puffing profiles as needed. Puff volume can be changed directly by adjusting the air flow meter. Puff time and heating coil activation time can be altered by changing the SVT and IPT constants in the program SVL.bs1 (Figure 2B, #5 & #6). If one were to want to de-synchronize the puffing time and the activation time, this could be done by splitting the SVT and IPT constants, e.g. SVT1 representing the time between the activation of the heating circuit and the valve circuit, SVT2 representing the time between the activation of the valve circuit and the inactivation of the heating circuit, and SVT3 representing the time between the inactivation of the heating circuit and the inactivation of the valve circuit, and likewise for IPT. While the 510 threaded base is common in many tank electronic cigarettes, it is not universal. A differently threaded base can be substituted if the user requires. If a square wave profile is not desired, replace either the air flow meter or solenoid valve with a continuous programable component to reshape the wave profile.
As electronic cigarette research progresses, the availability and accessibility of electronic cigarette smoking devices remains a hurdle. Cigarette smoking machines have been an integral part of tobacco product research as early as 1843 and today there are a variety of commercially available smoking machines for conventional cigarettes19,20. There are multiple established standard operating procedures for conventional cigarette smoking21. However, many conventional cigarette smoking devices proved incapable of accurately smoking electronic cigarettes due to the design differences between conventional and electronic cigarettes and differences within electronic cigarette brands and models, such as: diameter, PSI requirements, and the need for sensor or button based activation17. Currently there is a heterogenous commercial field of electronic cigarette smoking machines that includes devices including devices designed for direct aerosol extraction as well as air liquid interface exposure (such as Borgwalt22 and Vitrocell22,23). Despite the availability of commercial options, many groups, continue to use devices fabricated within their own laboratory for aerosol extraction 10,11,12,24,25,26. The motivations for this are varied. In some cases, researchers seek to better model human behavior10. Others are attempting to maintain continuity with previously published studies of cigarette smoke12. Still others directly cite the inaccessibility of commercial alternatives as a motivation for in laboratory fabrication24. These devices take many forms and, in many cases, use laboratory specific protocols. Unfortunately, the mechanisms, efficacy, and capabilities of these devices and protocols are often under-reported.
The first of the two sample applications presented above (Figure 3A) demonstrates the effects of conventional cigarette smoke and electronic cigarette aerosol on endothelial cell viability. As conventional cigarette smoke has been demonstrated to cause endothelial cell death and dysfunction9, it's reasonable to hypothesize that electronic cigarette aerosol would have a similar effect. To test this, we exposed human umbilical vein endothelial cells to nicotine equivalent levels of either conventional cigarette smoke extract or electronic cigarette aerosol extract for 24 h. While both conventional cigarette smoke and electronic cigarette aerosol cause statistically significant reductions in cell viability, the effect size of the electronic cigarette aerosol induced reduction is ~13% while the reduction after conventional cigarette smoke exposure nears 90%. While this supports the idea that electronic cigarettes are less harmful to the vascular system than conventional cigarettes, they are still not safe. The second of the two sample applications presented above (Figure 3B) demonstrates that electronic cigarette aerosol extracted into organic solvent can be separated into its components and analyzed via mass spectrometry. The component list generated provides information about the accuracy of labeling in electronic cigarette products, and highlights certain potentially harmful components such as acetyl propionyl (2,3-pentanedione)27. While the components identified in this experiment were not quantified, quantification can be performed by conventional analytical techniques such as those presented in CORESTA CRM8428.
Here, we have presented a programmable laboratory device capable of generating liquid extract from the conventional cigarette smoke or electronic cigarette aerosol. This device can accommodate a diverse array of product designs (such as the leading commercial brands of electronic cigarette) and the extraction process can be customized to user specifications. In this specific instance, we have demonstrated the use of generated extract in an endothelial cell viability assay; however, the extracts generated by this device could be applied to any type of single cell population as well as co-culture, explant, or other in vitro model. These extracts are compatible with a wide number of frequently used biological assays including reactive oxygen species detection, cell proliferation assays, and conventional immuno-staining. Moreover, the ability to break down the composition of electronic cigarette extract via gas-chromatography mass-spectrometry provides a starting point for detailed studies of individual aerosol components. Overall, this device provides an accessible entry point to electronic cigarette research.
The authors have nothing to disclose.
The authors acknowledge the assistance of Dr. Robert Dotson of the Tulane University Department of Cell and Molecular Biology for his assistance in editing the manuscript and Dr. James Bollinger of the Tulane University Department of Chemistry for his assistance with mass spectrometry protocol design. The authors further acknowledge the Tulane University Department of Cell and Molecular Biology and the Tulane University Department of Chemistry for their support and the use of space and equipment. This work was supported by a Tobacco Product Regulatory Science Research Fellowship to C. Anderson from the Tulane University School of Science and Engineering.
12 V AC/DC Wall Mount Adaptor | Digi-Key | T1099-P5P-ND | |
2.2 Ohm Resistors | Digi-Key | A105635-ND | Used in tandem to generate the 4.4 Ohm resistance in Figure 2A |
330 Ohm Resistors | Digi-Key | 330QBK-ND | |
510 Threaded Base | NJoy | N/A | Recovered by dismantalling a second generation NJoy electronic cigarette |
Acetic Acid, Glacial | Sigma-Aldritch | A6283 | |
Acetone (Chromatography Grade) | Sigma-Aldritch | 34850 | |
Basic Stamp Project Board | Digi-Key | 27112-ND | This board contains the BS1 Microcontroller, serial adaptor, power switch, and a barrel pin connector for the AC/DC Wall Mount Adaptor |
Basic Stamp USB to Serial Adapter | Digi-Key | 28030-ND | An optional component to allow the BS1 serial adaptor to communicate through USB |
Buchner Flask (Vacuum Flask) 250 mL | VWR | 10545-854 | |
Clear Tape | 3M | S-9783 | |
Clear Vinyl Tubing, 3/8" ID | Watts | 443064 | |
EGM-2 Endothelial Cell Culture Medium | Lonza | CC-3162 | |
Ethanol | Pharmco-Aaper | 111000200 | |
Flow Regulator | Dwyer | VFA-23-BV | |
Gas Chromatograph | Varian | 450-GC | |
Glass Syringe, 10 mL | Sigma-Aldritch | Z314552 | |
Glass Syringe, 10 µL | Hamilton | 80300 | |
High Vacuum Silicon Grease | Dow Corning | 146355D | |
Hose Clamp | Precision Brand | 35125 | |
Human Umbilical Vein Endothelial Cells | ATCC | PCS-100-013 | |
Mass Spectrometer | Varian | 300-MS | |
Midget Impinger | Chemglass | CG-1820-01 | |
Neutral Red | Sigma-Aldritch | N4638 | |
Paraffin Film | 3M | PM-992 | |
Plate Seal Roller | BioRad | MSR0001 | |
Plate Seal; Foil | Thermo | 276014 | |
Ring Stand 20" | American Educational Products | 7-G15-A | |
Solenoid Valve (normally open) | US Solid | USS2-00081 | |
Solid State Relay | Digi-Key | CLA279-ND | |
Stand Clamp | Eisco | CH0688 | |
Syringe Filter, PES, 0.22 um | Millipore | SLGP033RS | |
Syringe, 10 mL | BD Syringe | 309604 | |
Through Hole Stopper, Size 6 | VWR | 59581-287 | |
Vacuum Pump | KNF Neuberger | N86KTP |